Anti-carbamylated protein antibodies and the risk for arthritis

ABSTRACT

Antibodies against citrullinated protein antigens (ACPA) have shown their relevance for the diagnosis and possibly pathogenesis in arthritis. Described are means and methods for determining antibodies against homocitrulline-containing proteins or carbamylated proteins/peptides (anti-CarP) for the classification of individuals suffering from, or at risk of suffering from, arthritis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/120,946, filed Sep. 4, 2018, pending, which is a continuation of U.S.patent application Ser. No. 15/478,461, filed April 4, 2017, now U.S.Pat. No. 10,078,080, issued Sep. 18, 2018, which is a divisional of U.S.patent application Ser. No. 13/983,374, filed Feb. 27, 2014, now U.S.Pat. No. 9,632,084 issued Apr. 25, 2017, which is a national phase entryunder 35 U.S.C. § 371 of International Patent ApplicationPCT/NL2012/050056, filed Feb. 1, 2012, designating the United States ofAmerica and published in English as International Patent Publication WO2012/105838 A1 on Aug. 9, 2012, which claims the benefit under Article 8of the Patent Cooperation Treaty and under 35 U.S.C. § 119(e) toEuropean Patent Application Serial No. 11182399.3, filed Sep. 22, 2011,and to European Patent Application Serial No. 11153046.5, filed Feb. 2,2011, the disclosure of each of which is hereby incorporated herein inits entirety by this reference.

STATEMENT ACCORDING TO 37 C.F.R. § 1.821(c) or (e)—SEQUENCE LISTINGSUBMITTED AS A TXT AND PDF FILES

Pursuant to 37 C.F.R. § 1.821(c) or (e), files containing a TXT versionand a PDF version of the Sequence Listing have been submittedconcomitant with this application, the contents of which are herebyincorporated by reference.

TECHNICAL FIELD

The disclosure relates to the fields of post-translational modificationand arthritis. In particular, it relates to methods classifying samplesof individuals based on the detection of post-translationally modifiedproteins or peptides or antibodies specific for post-translationallymodified proteins or peptides in a sample containing a body fluid of theindividual.

BACKGROUND

There are over 100 different forms of arthritis. The most common form isosteoarthritis (degenerative joint disease). Osteoarthritis is mostcommonly the result of a trauma or of an infection of the joint, albeitthat there are also not readily identifiable causes. The latter areoften collectively referred to as “age-related osteoarthritis.” Otherforms of arthritis are, for example, rheumatoid arthritis, psoriaticarthritis, and related autoimmune diseases.

The major complaint by individuals who have arthritis is joint pain.Pain is often constant and may be localized to the joint affected. Thepain from arthritis is often the result of damage to the joint or theresult of the inflammation that occurs around the joint. Othercomplaints are pain as a result of muscle strains caused by forcefulmovements against stiff, painful joints and fatigue.

The diagnosis of patients with joint-associated pain is not easy as thecomplaints are typically vague and can be attributed to a variety ofdifferent causes, some of which are not arthritis. Indeed, many patientsthat first see a doctor with joint pain-associated complaints go intoremission and don't develop a chronic form of arthritis, whereas asignificant minority progress to develop Rheumatoid Arthritis (RA). Itis clear that individuals that go into remission spontaneously do notneed to receive treatment, whereas individuals that progress wouldbenefit considerably from early treatment. To discriminate between therespective groups, the field has developed a series of tests with whichthe diagnosis of arthritis can be made with more certainty. Such testspresently involve the screening of tissue samples and/or body fluidsamples for the presence of arthritis indicators therein. Suchindicators are, among others, determination of “chronic” inflammationindicators such as, for instance, certain chemokines, cytokines andother immune cell signaling factors; the determination of “accumulationof” active immune cells in joints, and/or the presence of the certainfactors in the blood, the most notable of which is rheumatoid factor.Recently, tests directed toward the detection of citrullinated proteinor peptides or antibodies specific for such citrullinated protein orpeptide have been developed as a useful tool for such tests. Theavailability of such tests has greatly improved the diagnosis ofindividuals suspected of having a form of arthritis. These tests alsoaid the clinician in giving a more accurate prognosis for the futuredevelopment of the disease to individuals suffering from arthritis.However, in spite of these developments, the diagnosis of arthritis orindividuals at risk thereof still leaves much to be desired.

For example, in the Netherlands, the recommendation to diagnose RA isbased on a probability score generated by the ACR/EULAR 2010 criteria.This criteria combines clinical features such as involvement of type andnumber of joints, presence or absence of the serological factors,rheumatoid factor and anti-CCP antibodies, presence or absence of acutephase proteins such as CRP, and duration of complaints. Patients thatare diagnosed with RA according to this protocol need to have more thansix points. The fact that only the clinical involvement and duration ofcomplaints are sufficient for a positive diagnosis indicates that thearthritis population is very heterogeneous and, in fact, often a wrongdiagnosis is made.

In the Netherlands and in other countries, tests for the typing ofarthritis or diagnoses of an individual at risk of developing arthritispresently include tests for the detection of antibodies specific forcitrullinated protein or peptide in samples of body fluids of suchpatients. These tests have led to the insight that arthritis patientscan be classified on the basis of positivity for antibodies directedtoward citrullinated proteins (ACPA). The identification of ACPA has hadan important impact on the understanding of RA.⁽¹⁾ Major differenceshave been observed when comparing ACPA-positive vs. ACPA-negative RApatients regarding genetic and environmental risk factors,⁽²⁾progression,⁽³⁾ remission,⁽⁴⁾ and response to treatment.⁽⁵⁾ Over therecent years, much more insight has been gained into the occurrence andetiopathology of ACPA-positive RA. However, much less information isavailable on ACPA-negative RA. In part, this is because it is relativelydifficult to identify or even subgroup these individuals as no goodassays are currently available. Interestingly, rituximab treatment hasbeen reported to also be beneficial in patients negative for RF andACPA.^((6, 7))

The post-translational modification of arginine residues into citrullineresidues by the PAD enzymes is the essential step to generate antigensfor ACPA.⁽¹⁾ Under physiological circumstances, this citrullination isimportant in tissues like hair and skin to generate layers of tissuethat are not-well connected.⁽⁸⁾ Also, in the nucleus, citrullinationplays a role in epigenetic regulation⁽⁹⁾ and condensation of chromatin,which is important, both in translation⁽⁸⁾ and in host defense againstpathogens.⁽¹⁰⁾ Under pathological conditions where cell death mayoverwhelm the phagocytic capacity, necrotic cell death may release PADinto the extracellular space, where higher calcium concentrations nowalso allow other host molecules to become citrullinated.⁽⁸⁾ Since manyof these molecules will be presented to the immune system as non-self,it can induce an antibody response in some individuals. Citrullinehighly resembles (FIG. 1) another post-translationally modified aminoacid called homocitrulline.⁽¹¹⁾ Homocitrulline is only one carbonlonger, but similar in structure.⁽¹¹⁾ Homocitrulline is generated from aLysine residue following an attack from cyanate, which exists in thebody in equilibrium with urea. Under physiological conditions, the ureaconcentration may be too low to allow extensive carbamylation (theprocess of changing lysine to homocitrulline). In conditions of renalfailure, the urea concentration increases and carbamylation can bereadily detected. However, most carbamylation is taking place duringinflammation when myeloperoxidase (MPO) is released fromneutrophils.⁽¹²⁾ This enzyme strongly shifts the equilibrium of ureatoward cyanate, now allowing more carbamylation to occur.⁽¹³⁾ It hasbeen shown recently that homocitrulline-containing proteins are presentin the RA joint and that this may affect T-cell triggering andautoantibody formation in animal models.^((11, 14)) Although highlysimilar, carbamylation differs from citrullination as, next to theirstructural difference, lysine and not arginine is modified. Therefore,homocitrulline will, by definition, be located at other positions inproteins than citrulline.

DISCLOSURE

Autoantibodies against carbamylated proteins have been found to bepresent in arthritis, and it has been determined that measurement ofsuch antibodies is useful in the diagnosis, prognosis, and management(e.g., treatment) of early arthritis and RA. Thus, provided is a methodfor classifying an individual that is suffering from, or at risk ofsuffering from, a form of arthritis, the method comprising determiningwhether a sample comprising a body fluid of the individual comprises ananti-Carbamylated Protein (anti-CarP) antibody.

An individual that is suffering from arthritis can be classified, on thebasis of anti-CarP antibodies, as low or high risk of developing a moresevere form of arthritis. Individuals with arthritis that are positivefor anti-CarP tend to develop a more severe form of arthritis thananti-CarP-negative individuals with arthritis, at least within any giventime period. The anti-CarP-positive individuals also tend to progressinto more severe forms more quickly when compared withanti-CarP-negative individuals. The method is thus preferably used toallocate an individual that is suffering from a form of arthritis to agroup with a lower or a higher than average risk for progressing to amore severe or chronic form of arthritis. In a preferred embodiment, theindividual is suffering from undifferentiated arthritis at presentation.In a preferred aspect of this embodiment, the more severe or chronicform is RA. In another preferred aspect of this embodiment, the moresevere or chronic form is juvenile arthritis, preferably juvenileidiopathic arthritis. In a preferred embodiment, the group with a lowerthan average risk is a group that has a higher than average incidence ofspontaneous remission of the arthritis complaints.

The at-risk population may be healthy individuals, patients sufferingfrom undifferentiated arthritis or arthralgia, autoantibody-positiveindividuals with joint complaints, autoantibody-positive individuals, orfamily members from patients with arthritis. “Juvenile idiopathicarthritis” is the term used for a subset of arthritis seen in childhood,which may be transient and self-limiting or chronic. Children withjuvenile idiopathic arthritis are considered an at-risk population, assome of these patients may develop a chronic form of arthritis.

The application shows that detection of anti-CarP antibodies is usefulin individuals that present with undifferentiated arthritis, arthralgia,and other joint complaints, and/or with juvenile arthritis. The presenceor absence of anti-CarP antibodies is predictive of the development ofRA or persistent arthritis later in life. This predictive power isobserved both in ACPA-negative and ACPA-positive subjects.

The method may be used to predict whether the individual is at risk ofdeveloping RA or persistent arthritis later in life.

For instance, human carbamylated fibrinogen can be used as a target foranti-CarP antibodies. Carbamylated fibrinogen is recognized by both IgGand IgA anti-CarP antibodies. Intact fibrinogen, or any of peptidesderived from carbamylated fibrinogen, can be used as targets in assaysto detect anti-CarP antibodies. The intact protein or any of thepeptides derived from fibrinogen can be used directly in the assay orcan be immobilized via an attached biotin group that will bind tostreptavidin-coated surfaces or can be used in any other way to detectanti-CarP antibodies. In a preferred embodiment, a method according tothe invention is provided, wherein the anti-CarP antibody is capable ofspecifically binding carbamylated fibrinogen. Throughout thedescription, the abbreviation “anti-Ca-Fib antibody” is used to indicatesuch antibody capable of specifically binding carbamylated fibrinogen.

Progression of disease in arthritis, for instance, progressive paincomplaints or progressive joint damage, is not a constant. Theprogression may be faster or slower for some period of time. Whenmention is made herein of a lower or higher risk of progression, this istypically compared to the average risk of progression within the groupof individuals that is studied.

Progression of diseases is at a group level established every year, sothat follow-up data can be analyzed using repeated measurement analysis.Yet, at the individual levels, positivity for anti-CarP, preferablyanti-Ca-Fib, is predictive for future progression. Currently, severalmethods including MRI, ultrasound and other techniques are available tomeasure progression of disease in the short term.

To assess whether a sample comprises anti-CarP antibodies, preferablyanti-Ca-Fib antibodies, a test for the presence of the antibodies isperformed. Such tests can involve, but are not limited to, ELISA and/orWestern blot, using one or several carbamylated proteins and/orpeptides. Commonly, though not necessarily, the result obtained for thesample is compared with a reference. The reference is typically theresult of a similar test, preferably the same test, performed on one ora number of healthy individuals (i.e., not known to suffer fromarthritis and not known to be at immediate risk of developingarthritis). The result of the sample can be directly compared with theresult of the reference, or the reference can be used to determine athreshold, below which any sample is to be judged a negative foranti-CarP, preferably anti-Ca-Fib, antibodies when below the thresholdor positive when above the threshold.

Further provided is a method for providing a prognosis for thedevelopment of arthritis to an individual suffering from arthritis, themethod comprising determining whether a sample comprising a body fluidof the individual comprises an anti-Carbamylated Protein (anti-CarP)antibody, and estimating the future severity of the arthritis based onthe detection of the anti-CarP antibody in the sample. In a preferredembodiment, the anti-CarP antibody is capable of specifically bindingcarbamylated fibrinogen (anti-Ca-Fib antibody). This estimation istypically accompanied by a time interval within which the more severeform or progression becomes apparent or not (see hereinabove).

One advantage of the classifications as indicated hereinbefore is thatthe groups of individuals have more homogeneous genetic profile withineach group than with other methods. Another advantage is that the groupsof individuals are more homogeneous in their response to (prophylactic)treatment. Since it has been shown that early aggressive treatment isbeneficial,^((18, 19)) provided are methods for arthritis treatment ofindividuals suffering from, or at risk of suffering from, arthritis, themethod comprising an arthritis diagnosis of the individual wherein thediagnosis comprises a method for determining an anti-CarP antibody,preferably an anti-Ca-Fib antibody, in a sample comprising a body fluidof the individual. Preferably, the sample was determined to contain ananti-CarP antibody, preferably an anti-Ca-Fib antibody. A more stringenttreatment of the anti-CarP or anti-Ca-Fib-positive individual isbeneficial to the patient. The treatment is typically a therapeutictreatment given to a patient that was diagnosed with arthritis prior toreceiving the treatment. Further provided is a method of treating anindividual suffering from arthritis with an arthritis medication and/ortreatment, the method characterized in that the individual was diagnosedwith a method hereof prior to the treatment. Preferably, the individualwas diagnosed as suffering from arthritis with a method for classifyingindividuals suffering from arthritis of the invention, prior toreceiving the arthritis medication and/or treatment. Further provided isa method for the prophylactic treatment of an individual at risk ofdeveloping arthritis with an arthritis medication and/or treatment, themethod characterized in that the individual was diagnosed being at riskof developing arthritis with a method for classifying individuals of theinvention, prior to receiving the arthritis medication and/or treatment.Treatment, in this case, prophylactic, can also be given to individualsthat are classified to be at risk of developing the disease in the nearfuture, typically within one year of classification. Such prophylactictreatments are capable of at least postponing the onset of the diseaseand/or reducing the severity of the disease.

The methods used to classify arthritis patients or individuals at riskof developing arthritis typically involve a number of different tests.Such tests may also be combined with a method of the invention to arriveat a more accurate assessment of the classification. To this end, theinvention further provides means and methods to further classifyindividuals that suffer from arthritis or that are suspected/at risk ofhaving arthritis. In a preferred embodiment, a method of the inventionis combined with a test for Anti-Citrullinated Protein Antibodies(ACPA).

The ACPA test has been used for quite some time (reviewed, among others,in Venrooij et al. “Anticitrullinated protein/peptide antibody and itsrole in the diagnosis and prognosis of early rheumatoid arthritis,” TheNetherlands Journal of Medicine, 2002, 60:383-388; and L. Klareskog, J.Ronnelid, K. Lundberg, L. Padyukov, and L. Alfredsson, “Immunity tocitrullinated proteins in rheumatoid arthritis,” Annu. Rev. Immunol.2008, 26:651-75.

Many different citrullinated proteins or peptides can be used in teststo detect anti-citrullinated protein antibodies (ACPA). Citrullinatedfilaggrin has been used to detect the so-called anti-filaggrinantibodies (AFA). In first attempts to find suitable substrates for RAauto-antibodies, a number of linear peptides containing one citrullineresidue were developed. These citrullinated peptides were specificallyrecognized by the RA auto-antibodies and, more important, theirarginine-containing counterparts were not. However, most peptidesreacted with only 30 to 45% of the RA sera, although more than 75% of RAsera reacted with at least one of a total of nine peptides tested(Schellekens et al. (1998), J. Clin. Invest. 101:271-281). Althoughlinear peptides may be used, it has been found that cyclic peptidesrender the tests more sensitive. Tests that include cyclic citrullinatedprotein/peptide are typically referred to as CCP tests. The CCP1 testwas already sensitive, but it has been found that a novel selection ofcyclic citrullinated protein/peptides with improved immune recognitionproperties has increased the sensitivity of the CCP test to at least80%. This latter test is typically referred to as the CCP2 test. Thus,in one embodiment hereof, the method for detecting ACPA comprisesdetecting anti-cyclic citrullinated protein/peptide in the sample.Preferably, the method for detecting ACPA is a CCP2 test as described inG. J. Pruijn, A. Wiik, and W. J. van Venrooij, “The use of citrullinatedpeptides and proteins for the diagnosis of rheumatoid arthritis,Arthritis Res. Ther. 2010, 12(1):203, Epub. 2010 Feb. 15; and F. A. vanGaalen, H. Visser, and T. W. Huizinga, “Review and a comparison of thediagnostic accuracy and prognostic value of the first and secondanti-cyclic citrullinated peptides (CCP1 and CCP2) autoantibody testsfor rheumatoid arthritis,” Ann. Rheum. Dis., October 2005, 64(10):1510-2.

As is the case for ACPA, also for an anti-CarP antibody in principle,many different carbamylated proteins or peptides can be used in tests todetect anti-carbamylated protein antibodies. In a preferred embodiment,the carbamylated protein or peptide is a cyclic peptide. A good sourceof carbamylated proteins is carbamylated fetal calf serum. Serumproteins from other species are, however, also suitable. Followingoptimization, human proteins might also be used. For reasons ofinevitable background, human serum cannot be used in carbamylated formwithout extensive depletion of Ig. In a preferred embodiment, thecarbamylated protein is fibrinogen, preferably human fibrinogen.

A peptide for use in a method hereof, be it for the detection of ACPA ingeneral or of anti-CarP antibodies or of anti-Ca-Fib antibodies, istypically a peptide of between 6 and 50 amino acids. Preferably, thepeptide is a peptide of between 12 and 30 amino acids, more preferablyof between 18 and 22 amino acids, most preferably of about 21 aminoacids. The mentioned ranges include the number mentioned, i.e., a rangeof between 12 and 30 amino acids includes peptides of 12 and 30 aminoacids, respectively. The peptide may or may not be a cyclic peptidedepending on the sensitivity and/or specificity than the comparablelinear peptide. Circular peptides can be generated in any molecularcomposition as to generate the cyclic nature. Any method may be used tocouple peptides and or proteins in carbamylated, citrullinated or nativeform to plates and/or beads, being either direct coating or usingbiotin-streptavidin or any other available coating method.

In a preferred embodiment, such peptide for use in a method of theinvention is a peptide derived from human fibrinogen. The peptidepreferably comprises a contiguous amino acid of between 12 and 30 aminoacids, more preferably of between 18 and 22 amino acids, most preferablyof about 21 amino acids present in the amino acid sequence of any one offibrinogen alpha (FIG. 14), fibrinogen beta (FIG. 15) or fibrinogengamma (FIG. 16). In a more preferred embodiment, the peptide is any oneof the peptides depicted in Table I, Table II, or Table III.

Further provided is, as indicated hereinabove, a method for classifyingan individual that is suffering from, or at risk of suffering from, aform of arthritis, the method comprising determining whether a samplecomprising a body fluid of the individual comprises an anti-CarPantibody, preferably an anti-Ca-Fib antibody, and determining whetherthe sample comprises Anti-Citrullinated Protein Antibodies (ACPA) andclassifying the individual on the basis of the detection of theanti-CarP antibody and/or the ACPA.

The anti-CarP antibody, the anti-Ca-Fib antibody, and ACPA can be of anyimmunoglobulin isotype. The art typically focuses on one or more of theIgG subclasses. In the present invention, it has been found that thelevel of an anti-CarP antibody, anti-Ca-Fib antibody and/or ACPA of boththe Ig-subtype A (IgA) and the Ig-subtype G (IgG) in a sample comprisingbody fluid of the individual is predictive for clinical outcome measuressuch as joint destruction. It has also been found that whereas a samplecan be negative for anti-CarP or anti-Ca-Fib IgG, it can be positive foranti-CarP or anti-Ca-Fib IgA and vice versa. Thus, in a preferredembodiment of a method hereof, the method comprises determining whethera sample comprising a body fluid of the individual comprises ananti-CarP and/or anti-Ca-Fib antibody of Ig-subtype IgA or an anti-CarPand/or anti-Ca-Fib antibody of Ig-subtype IgG, or both, and whereindetection of the IgA and/or IgG anti-CarP and/or anti-Ca-Fib antibodyindicates that the individual is suffering from, or at risk of sufferingfrom, arthritis. In a preferred embodiment, the method further comprisesdetermining whether a sample comprising a body fluid of the individualcomprises ACPA. As mentioned hereinabove, a method of the invention isparticularly useful in subdividing the heterogeneous group ofACPA-negative individuals. Using a method hereof, this ACPA-negativegroup can be divided into a group that is anti-CarP and/or anti-Ca-Fibantibody positive and that can be classified as suffering from, or atrisk of suffering from, arthritis, and a group that is anti-CarP and/oranti-Ca-Fib antibody negative that can be classified as not sufferingfrom arthritis or not at risk of suffering from arthritis. Thisdetermination can also be done on an individual basis. Thus, a methodhereof may further comprise determining whether a sample comprising abody fluid of the individual comprises Anti-Citrullinated ProteinAntibodies (ACPA) and wherein the level of the ACPA in the sample isbelow the detection limit and or cut-off of positivity.

A sample of an individual tested for the presence of an anti-CarPantibody, preferably of isotype IgA or of isotype IgG, or both, and ACPAcan be classified on the basis of the result as an:

-   -   a) an anti-CarP and/or anti-Ca-Fib antibody positive,        ACPA-negative sample;    -   b) an anti-CarP and/or anti-Ca-Fib antibody positive,        ACPA-positive sample;    -   c) an anti-CarP and/or anti-Ca-Fib antibody negative,        ACPA-positive; and    -   d) an anti-CarP and/or anti-Ca-Fib antibody negative,        ACPA-negative sample.

Results a), b) and c) classify the sample as a sample of an individualthat is at high risk to be currently suffering from, or at risk ofdeveloping, arthritis. Result d) classifies the sample as a sample of anindividual that has a low risk to be currently suffering from ordeveloping arthritis. In the event that the individual presented withundifferentiated arthritis, then classifying the sample in group d)indicates that the individual has a high chance of spontaneousremission. Such an individual is not expected to benefit on the longterm from an anti-arthritis treatment. The invention further provides amethod for typing a sample comprising a body fluid of an individualsuffering from undifferentiated arthritis, the method comprisingdetermining whether the sample comprises an anti-CarP antibody,preferably an anti-Ca-Fib antibody, and typing the sample as derivedfrom an individual that has a higher than average chance of spontaneousremission of the arthritis when the sample was determined to be negativefor the anti-CarP antibody, preferably the anti-Ca-Fib antibody, andtyping the sample as derived from an individual that has a higher thanaverage chance of having or progressing to RA when the sample wasdetermined to be positive for the anti-CarP antibody, preferably theanti-Ca-Fib antibody. The higher than average chance is typicallyarrived at by comparison of the chance with the average chance arrivedat for a number of unselected individuals presenting withundifferentiated arthritis.

In another aspect, provided is a method for estimating the severity ofarthritis for an individual that is suffering from a form of arthritisand has an increased risk of developing a more severe form of arthritis,the method comprising determining whether a sample comprising a bodyfluid of the selected individual comprisesanti-homocitrulline-Containing Protein antibodies (an anti-CarPantibody), preferably anti-homocitrulline-Containing Fibrinogenantibodies (an anti-Ca-Fib antibody), and estimating the severity of thearthritis based on the detection of the anti-CarP antibodies,preferably, the anti-Ca-Fib antibody in the sample.

In yet another aspect, provided is a method for providing a prognosisfor the development of arthritis to an individual suffering fromarthritis, the method comprising determining whether a sample comprisinga body fluid of the individual comprises an anti-CarP antibody,preferably an anti-Ca-Fib antibody, and estimating the future severityof arthritis based on the detection of the anti-CarP antibody,preferably an anti-Ca-Fib antibody, in the sample. The method ispreferably combined with a method for determining ACPA is the sample andthe combined result of the anti-CarP and/or anti-Ca-Fib antibody testand the ACPA test is used to estimate the future severity of arthritisfor the individual.

The sample to be tested for the presence of an anti-CarP antibody,preferably an anti-Ca-Fib antibody, and/or ACPA can, in principle, beany type of sample as long as it contains body fluid of the individual.Typically, however, the sample is a sample of body fluid. In a preferredembodiment, the sample comprising body fluid is a serum sample or asynovial fluid sample.

Arthritis is, as mentioned hereinabove, a complex disease and manydifferent forms of arthritis have presently been identified. Thearthritis that the individual is suffering from or at risk of sufferingfrom is preferably an arthritis selected from rheumatoid arthritis,juvenile arthritis, psoriatic arthritis, osteoarthritis, polymyalgiarheumatica, ankylosing spondylitis, reactive arthritis, gout,pseudogout, autoimmune arthritis, systemic lupus erythematosus,polymyositis, fibromyalgia, Lyme disease, undifferentiated arthritis,non-rheumatoid arthritis or spondyloarthropathy. More preferably, thearthritis that the individual is suffering from or at risk of sufferingfrom is an arthritis selected from rheumatoid arthritis, psoriaticarthritis, osteoarthritis, polymyalgia rheumatica, ankylosingspondylitis, reactive arthritis, gout, pseudogout, autoimmune arthritis,systemic lupus erythematosus, polymyositis, fibromyalgia, Lyme disease,undifferentiated arthritis, non-rheumatoid arthritis orspondyloarthropathy. Preferably, the arthritis is selected fromrheumatoid arthritis, juvenile arthritis, more preferably juvenileidiopathic arthritis, or undifferentiated arthritis. More preferably,the arthritis is selected from rheumatoid arthritis or undifferentiatedarthritis.

Further provided is a method for typing the arthritis of an individualsuffering from a form of arthritis, the method comprising determiningwhether a sample comprising a body fluid of the individual comprises ananti-Carbamylated Protein (anti-CarP) antibody, preferably ananti-Carbamylated Fibrinogen (anti-Ca-Fib) antibody. The arthritis canbe typed on the basis of the detected presence or absence of theanti-CarP and/or anti-Ca-Fib antibody. A sample in which the anti-CarPantibody, preferably the anti-Ca-Fib antibody, is detected is morelikely to be derived from an individual with rheumatoid arthritis. Thus,this method can be used alone, or in combination with another test forRA to assess the likelihood that the individual is suffering from RA. Ina preferred embodiment, the further test comprises a test for thepresence of ACPA, preferably a CCP2 test and or a test for RheumatoidFactor.

Shown herein is that anti-CarP antibodies can be detected inasymptomatic, healthy individuals before RA development. Thus, detectionof anti-CarP antibodies are also useful in the healthy population forthe early identification of patients at risk to develop RA or persistentarthritis later in life. In addition, the detection of anti-CarPantibodies is useful to identify persons that would benefit from earlytreatment, preferably before they fulfill the current classification forRA.

In a further aspect, provided is a method for determining whether anindividual is at risk of developing or suffering from a form ofarthritis, and wherein the individual was not known to suffer, orpreferably not suffering from, arthritis at the time that the samplecomprising body fluid was collected, the method comprising determiningwhether a sample comprising a body fluid of the individual comprises anAnti-Carbamylated Protein (anti-CarP) antibody. In a preferredembodiment, a method hereof is provided wherein the anti-CarP antibodyis an Anti-Carbamylated Fibrinogen (anti-Ca-Fib) antibody. A sample inwhich the anti-CarP antibody, preferably the anti-Ca-Fib antibody, isdetected is likely to be derived from an individual that is at risk ofsuffering from or developing arthritis, in particular RA, in the nearfuture, particularly within five years from the date of samplecollection, particularly within three years and more particularly withinone and a half years from the date of sample collection. This method canbe used alone, or in combination with, another test for RA to assess therisk. In a preferred embodiment, the further test comprises a test forthe presence of ACPA, preferably a CCP2 test and/or a test forRheumatoid Factor.

In another aspect, provided is a kit for the detection of anti-CarPantibodies, preferably anti-Ca-Fib antibodies, in a body fluid of anindividual, the kit comprising a carbamylated protein or peptide.Preferably, the carbamylated protein or peptide is carbamylatedfibrinogen or a fibrinogen-derived peptide. In a preferred embodiment,the protein or peptide is a carbamylated protein or peptide as indicatedhereinabove. In particular, the kit comprises at least one peptidecomprising a contiguous amino acid of between 12 and 30 amino acids,more preferably of between 18 and 22 amino acids, most preferably ofabout 21 amino acids, present in the amino acid sequence of any one offibrinogen alpha (FIG. 14), fibrinogen beta (FIG. 15) or fibrinogengamma (FIG. 16). In a more preferred embodiment, the kit comprises atleast one of the peptides depicted in Table I, Table II, or Table III.

As also mentioned hereinabove, in a preferred embodiment, the kitfurther comprises an anti-human IgG antibody and/or an anti-human IgAantibody. Preferably, the anti-human Ig antibody comprises a label thatcan be detected. Non-limiting examples of such labels are a directlabeling with HRP or AP. Alternatively, the kit preferably furthercomprises another antibody, which antibody is specific for theanti-human Ig antibody used. In this embodiment, another antibodycomprises a label that can be detected, such as biotin or DIG. Thisnesting can, of course, be continued with label being present on thelast and/or one or more (earlier) antibodies. In a preferred embodiment,the anti-human Ig antibody comprises an anti-human IgA antibody. In amore preferred embodiment, the kit comprises an anti-human IgA antibodyand an anti-human IgG antibody. The anti-human Ig antibody, as indicatedhereinabove, is typically a full antibody; however, a fragment thatcontains the antigen binding site is also within the use of the term“antibody.” Similarly, there are presently a great variety of differentbinding proteins or peptides available that can be tailored tospecifically bind to human Ig. Such human Ig-specific binding proteinsor peptides are equivalents of an anti-human Ig antibody as definedherein. Similarly, the one or more further antibodies in a nestingsetting can be replaced with binding proteins and/or peptides thatspecifically bind the earlier antibody and/or binding protein/peptide inthe nesting tree.

In a particularly preferred embodiment, the kit further comprises acitrullinated protein or peptide. In a preferred embodiment, the proteinor peptide is a citrullinated protein or peptide as indicatedhereinabove. As also mentioned hereinabove, the citrullinated protein orpeptide is preferably a protein or cyclic peptide. Preferably, the kitcomprises a cyclic citrullinated peptide of a CCP1 or CCP2 test.Preferably, the kit comprises all cyclic peptides of a CCP test,preferably a CCP2 test.

A method hereof is, as mentioned hereinabove, preferably combined withanother arthritis classifier test. Thus, in a preferred embodiment of amethod hereof, the method further comprises determining a further factoras an arthritis classifier for the individual. Preferably, the furtherfactor comprises determining ACPA, rheumatoid factor, C-reactiveprotein, and/or erythrocyte sedimentation rate.

Carbamylation is defined herein as the process of providing a protein orpeptide with a modification that generates a homocitrulline residue. Inthis document where reference is made to a “carbamylated protein orpeptide or collection thereof,” reference is made to the protein orpeptide having a homocitrulline modification, or a collection ofproteins or peptide having homocitrulline modifications.

Further provided is an anti-CarP antibody, preferably an anti-Ca-Fibantibody, and ACPA for use in determining whether an individual issuffering from, or at risk of suffering from, a form of arthritis. Theinvention further provides an anti-CarP antibody, preferably ananti-Ca-Fib antibody, and ACPA for use in the classification ofarthritis, the determining and/or classification being a determining orclassification as indicated hereinabove.

Further provided is a method for determining whether an individual thatis suffering from a form of arthritis has an increased risk ofdeveloping a more severe form of arthritis, the method comprisingselecting an individual that is suffering from arthritis but that doesnot have the most severe form of arthritis and determining whether asample comprising a body fluid of the selected individual comprises ananti-Carbamylated Protein Antibody (an anti-CarP antibody), whereindetection of the anti-CarP indicates that the individual has increasedrisk of developing a more severe form of arthritis. In a preferredembodiment, a method according to the invention is provided, wherein theanti-CarP antibody is an anti-Carbamylated Fibrinogen Antibody (ananti-Ca-Fib antibody).

In this document where reference is made to “detection, determination orother assessment” of an anti-CarP antibody, an anti-Ca-Fib antibody oran ACPA, the reference includes that more than one anti-Carp antibody,anti-Ca-Fib antibody and/or more than one ACPA is detected, determinedor otherwise assessed. Anti-CarP or anti-Ca-Fib antibody can recognize ahomocitrulline modification per se or, more typically, recognize themodification in the context of one or more of the amino acids of theprotein or peptide in the immediate vicinity of the homocitrullinemodification. A method or kit of the invention is more accurate whenanti-CarP antibodies and/or anti-Ca-Fib antibodies of more than onespecificity is detected, determined or otherwise assessed. A method ofthe invention thus preferably comprises determining two or more, andpreferably three or more, more preferably five or more, most preferablyat least seven anti-CarP and/or anti-Ca-Fib antibodies in the sample.Similarly, a kit of the invention preferably comprises two or more, andpreferably three or more, more preferably five or more, most preferablyat least seven carbamylated proteins and/or peptides, preferablyfibrinogen and/or fibrinogen-derived peptides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A through 1C: Development of a novel and specific assay for thedetection of anti-CarP antibodies. (FIG. 1A) Dose response curves of theanti-CarP antibody-positive standard on carbamylated FCS and native FCSin ELISA. (FIG. 1B) Inhibition studies where anti-CarP antibody bindingto ELISA plates coated with Ca-FCS was inhibited using pre-incubationswith fluid-phase inhibitors as indicated. Only Ca-FCS inhibited thebinding of anti-CarP antibodies. (FIG. 1C) Coomassie staining showingequal loading of Ca-FCS and FCS, and Western blot showing a positivestaining of Ca-FCS-loaded lanes and not FCS-loaded lanes by a serumsample of an anti-CarP-positive sample and not by a negative sample.

FIG. 2: Anti-CarP IgG and IgA antibodies are present in RA sera. ELISAwas performed for the detection of anti-CarP IgG and IgA in sets of seraof healthy controls and RA patients. A cut-off was established using themean plus two times the standard deviation of the healthy controls.Shown is the titer expressed as arbitrary units per ml followingcalculation based on the standard curve. Below the graph, both thenumber of samples tested and the percentage positivity is indicated.

FIG. 3: Anti-CarP antibodies and ACPA are two separate autoantibodysystems. Pie charts showing the percentages of RA patients positive andnegative for ACPA and/or anti-CarP antibodies.

FIG. 4: Anti-CarP IgA antibodies are associated with the conversion ofundifferentiated arthritis (UA) to RA. Sera of patients that presentedwith UA at baseline were measured for anti-CarP IgG and IgA antibodiesand analyzed for their conversion toward RA at the one-year follow-up.Data shown are also split up on the basis of ACPA positivity. IgAanti-CarP antibodies associate strongly with development of RA from a UApopulation.

FIG. 5: Anti-CarP IgA antibodies are associated with more severeradiological progression in RA. The extent and rate of joint destructionwere analyzed in the RA patients split up on the basis of positivity forACPA and anti-CarP antibodies. Positivity for anti-CarP IgA antibodiesis associated with a more severe radiological damage in bothACPA-negative and ACPA-positive RA patients.

FIGS. 6A and 6B: Illustration of citrullination and carbamylation.Citrullination and carbamylation occur on different amino acids viadifferent mechanisms, but yield similar end-products.

FIG. 7: Antibodies against carbamylated proteins are present in sera ofRA patients. The reactivity of IgG (Panels A and B) and IgA (Panels Dand E) from sera of healthy controls (NHS) or RA patients (RA) to wellscoated with non-modified FCS (FCS) or carbamylated FCS (Ca-FCS) isdepicted. Data expressed as absorbance at 415 nm. In Panels C and F,absorbance units of FCS were subtracted from the absorbance units ofCa-FCS, representing the specific anti-carbamylated protein response.

FIG. 8: Anti-CarP antibodies and ACPA are two separate autoantibodysystems. Panel A depicts IgG reactivity of 76 sera from RA patientstoward several forms of a Fib peptide. Panels B and C depict binding toCa-FCS or Ci-FCS was inhibited using pre-incubations with fluid-phaseinhibitors. Panel D shows that FCS, Ca-FCS and Ci-FCS were separated bySDS-page gels and blotted. The presence of antibodies reactive toproteins on the blots was analyzed by incubating these blots with eitheranti-CarP-positive ACPA-negative and anti-CarP-negative ACPA-positivesera.

FIG. 9: Anti-CarP antibodies bind to Ca-Fib via variable domains. PanelA: IgG reactivity against Fib, Ci-Fib and Ca-Fib of 54 healthy controlsand 214 RA patients was analyzed by ELISA. Panel B: Specificity ofanti-Ca-Fib reactivity was confirmed using inhibition studies. Onesample is shown, where data are expressed relative to inhibition withPBS. Panel C: The molecular nature of purified IgG and F(ab′)2 wasconfirmed by Coomassie-stained SDS page gel. Panel D: F(ab′)2 fragmentswere generated from purified IgG of two anti-CarP-positive patients andtwo negative controls. Only F(ab′)2 from patients reacted with Ci-Fiband Ca-Fib. Panel E: Inhibition experiments also confirm that F(ab′)2are not necessarily cross-reactive between Ci-Fib and Ca-Fib.

FIG. 10: Anti-CarP IgG and IgA antibodies are present in RA sera. PanelsA and B: Dose response curves of the anti-CarP antibody-positivestandard (IgG and IgA) on Ca-FCS and FCS in ELISA. Panels C and D: ELISAwas performed for the detection of anti-CarP IgG and IgA in sera ofhealthy controls (NHS) and RA patients. A cut-off was established usingthe mean plus two times the standard deviation of the healthy controlsas described in the methods. Reactivity is depicted as arbitrary unitsper mL. The number of samples tested and the % positivity is indicatedbelow the graph. Panels E and F: Pie charts showing the % of RA patientspositive and negative for anti-CCP2 and/or anti-CarP antibodies. PanelsG and H: Pie charts showing the % of anti-CarP IgG- or IgA-positivepatients negative for anti-CCP2.

FIG. 11: Anti-CarP IgG antibodies are associated with a more severeradiological progression in ACPA-negative RA. The extent and rate ofjoint destruction were analyzed in all RA patients included, or analyzedseparately for ACPA-negative or ACPA-positive subgroups (FIG. 13). Theseverity of joint destruction is depicted as median Sharp/van der Heijdescore (SHS) on the Y-axis and the follow-up years on the X-axis. Thepatient number is listed for each time point below the X-axis.Radiological progression for the anti-CCP2-negative RA patients isshown. The P-value is derived from the analysis model as described inthe methods section.

FIGS. 12A through 12C: Anti-CarP IgG antibodies are associated with theconversion of pre-disease to RA. FIG. 12A: Sera of patients thatpresented with Undifferentiated Arthritis (UA) at baseline were measuredfor anti-CarP IgG antibodies and analyzed for their conversion toward RAat 1 year follow-up. Data shown are also split up on the basis of ACPApositivity. Anti-CarP antibodies associate strongly with development ofRA from a UA population. FIG. 12B: Also in sera of patients thatpresented with arthralgia at baseline, anti-CarP antibodies arepredictive for development of RA. FIG. 12C: Sera of healthy persons thatdo not develop RA or that do develop RA later in life are compared foranti-CarP positivity. The presence of anti-CarP is associated withfuture development of RA.

FIG. 13: Anti-CarP IgA antibodies are associated with more severeradiological progression in RA. The extent and rate of joint destructionwere analyzed in the RA patients split up on the basis of positivity forACPA and anti-CarP antibodies. Positivity for anti-CarP IgG antibodiesis associated with a more severe radiological damage in ACPA-negative RApatients.

FIG. 14: Amino Acid Sequence of Fibrinogen alpha (SEQ ID NO:1).

FIG. 15: Amino Acid Sequence of Fibrinogen beta (SEQ ID NO:2).

FIG. 16: Amino Acid Sequence of Fibrinogen gamma (SEQ ID NO:3).

FIG. 17: Anti-CarP antibodies are present in sera of patients sufferingfrom juvenile arthritis. ELISA was performed for the detection ofanti-CarP IgG in sera of healthy children (Ctr) and in sera of patientssuffering from juvenile arthritis. A cut-off was established using themean plus two times the standard deviation of the healthy controls asdescribed in the methods. Reactivity is depicted as arbitrary units permL. The number of samples tested and the % positivity is indicated belowthe graph.

DETAILED DESCRIPTION EXAMPLES Example 1 Materials and Methods Generationof Antigens

As a source of antigens, we have used fetal calf serum (FCS) (Bodinco,batch No. 212-192909). This was carbamylated, citrullinated or employedas an unmodified source.

Carbamylated FCS (Ca-FCS) was generated by diluting FCS in bidest to 4mg/ml. Potassium cyanate (sigma, Cat. No. 215074) was added at 80 mg/ml.Following incubation at 37° C. for 12 hours, the sample was extensivelydialyzed against bidest.

As a control, citrullinated FCS (Ci FCS). For this purpose, 50 μl FCS(24 mg/ml) with 24 μl 0.5 M Tris-HCl pH 7.6+15 μl 0.125 M CaCl₂+31 μlPAD4 (Sigma P1584) was incubated for 24 hours at 37° C.

Detection of Anti-CarP Antibodies by ELISA

Non-modified FCS and Ca-FCS were coated at 10 μg/ml (diluted in pH 9.60.1 M carbonate-bicarbonate buffer) 50 μl on Nunc immunoplates (ThermoScientific, Cat. No. 430341), overnight at 4° C. Following washing forfour times in phosphate-buffered saline (PBS) containing 0.05% TWEEN®(Sigma, Cat. No. 27,434-8) (PT), the plates were blocked by incubating100 μl PBS/1% bovine serum albumin (BSA) (Sigma, Cat. No. A2153) for 1hour at 37° C. Following additional washing, the sera were incubated in50 μl at a 1/50 (in PBS/0.05% TWEEN®/1% BSA buffer (PTB)) to both FCS-and Ca-FCS-coated wells and incubated at 37° C. for 1 hour. Serialdilutions of a standard serum (diluted in PTB) were incubated onCa-FCS-coated wells. Following washing, bound human IgG or IgA wasdetected by incubating the wells with 50 μl 1/5000 diluted (in PTB)rabbit anti-human IgG antibody (Dako, Cat. No. A0423) or 1/1000 diluted(in PTB) rabbit anti-human IgA antibody (Dako, Cat. No. A0262) incubatedat 37° C. for 1 hour. Following washing, wells were incubated at 37° C.for 1 hour with 50 μl 1/2000 diluted (in PTB) goat anti-rabbit IgGHRP-labeled antibody (Dako, Cat. No. P0448). Following the lastwashings, HRP enzyme activity was visualized by incubating 50 μl2,2′Azino-bis-(3-ethylbenzo-thiazole-6-sulfonic acid) diammonium salt(ABTS) and H₂O₂, measuring absorbance at 415 nm on a standard ELISAreader.

Detection of Anti-CarP Antibodies by Western Blot

Both FCS and Ca-FCS were loaded onto regular 10% sodium dodecyl sulfate(SDS)-polyacrylamide gels and transferred onto Hybond-C Extra membranes(Amersham, Diegem, Belgium). Blots were then incubated in blockingbuffer (3% ELK Milk/PBS/0.05% TWEEN®) 1 hour at RT, following washingwith PBS/0.05% TWEEN®. The blots were subsequently incubated with 5 mlserum 1:500 diluted in blocking buffer for 1 hour at RT. After threewashes with PBS/0.05% TWEEN®, blots were incubated with 3 ml horseradishperoxidase conjugated rabbit anti-human IgG (DAKO, Heverlee, Belgium)1:50,000 diluted in blocking buffer for 1 hour at RT. Next, blots werewashed and bound antibodies were visualized using enhancedchemiluminescence (ECL; Amersham). Equal protein loading was verifiedusing Coomassie Brilliant Blue (Bio-Rad, Veenendaal, The Netherlands).

Sera and Synovial Fluids

The sera analyzed were from patients participating in the Leiden EarlyArthritis Clinic (EAC) cohort. The Leiden EAC is an inception cohort ofpatients with recent-onset arthritis (symptoms duration <2 years) thatwas started at the Department of Rheumatology of the Leiden UniversityMedical Center in 1993.⁽¹⁵⁾ All RA patients fulfilled the AmericanCollege of Rheumatology (formerly the American Rheumatism Association)1987 revised criteria for the classification of RA⁽¹⁶⁾ within one yearof follow-up (EAC cohort). A total of 1007 patients were analyzed, ofwhich 582 were diagnosed as RA and 425 as UA, of which 151 developed RAon follow-up. These patient samples were compared to 280 healthy controlsamples also derived from the Leiden area. An additional set of pairedserum/synovial fluid of RA patients was analyzed. The protocols wereapproved by the relevant local ethics committee and all participantsprovided informed consent.

ELISA for the Detection of ACPA

Total IgG anti-CCP2, as a measure of ACPA, was measured in seracollected at baseline by enzyme-linked immunosorbent assay (ELISA)(Immunoscan RA Mark 2; Eurodiagnostica, Arnhem, The Netherlands).Samples with a value above 25 units/ml were considered positiveaccording to the manufacturer's instructions. Individuals withantibodies against CCP2 were considered ACPA-positive.

ELISA for the Detection of Anti-CaFib Antibodies

Non-modified Fib and Ca-Fib were coated at 20 μg/ml in 50 μl (diluted inpH 9.0 PBS) on Nunc Maxisorp plates ON. Following washing in PBS TWEEN®,the plates were blocked by incubating 200 μl pH 9.0 PBS/2% BSA for 2hours at 4° C. Following additional washing, the wells were incubatedwith 50 μl serum at a 1/50 dilution in RIA buffer (10 mM Tris pH 7.6;350 mM NaCl; 1% TritonX; 0.5% Na-deoxycholate; 0.1% SDS) (Sigma) on icefor 3 hours. All subsequent incubations are performed in RIA buffer. Asa standard, serial dilutions of a pool of positive sera were used. HumanIgG was detected using HRP-labeled rabbit anti human IgG antibody (DAKO)incubated on ice for 2 hours. Following the last washings, HRP enzymeactivity was visualized using ABTS. We transformed the absorbance on Fiband Ca-Fib to aU/mL. We established the cut-off for a positive responseas the mean plus 2× the standard deviation of the specific anti-CarPreactivity of the healthy controls.

We analyzed 67 sera of healthy children and 110 sera of patientssuffering from juvenile arthritis.

Statistics

Data were analyzed using the Statistical Package for the Social Sciences(SPSS) 17.0 using logistic regression. P-values below 0.05 wereconsidered to be statistically significant.

Example 2 Materials and Methods Patient and Control Sera

The sera analyzed were from patients participating in the Leiden EarlyArthritis Clinic (EAC) cohort. The Leiden EAC is an inception cohort ofpatients with recent-onset arthritis (symptoms duration <2 years) thatwas started at the Department of Rheumatology of the Leiden UniversityMedical Center in 1993.⁽³⁹⁾ All RA patients fulfilled the AmericanCollege of Rheumatology (formerly the American Rheumatism Association)1987 revised criteria for the classification of RA⁽⁴⁰⁾ within 1 year offollow-up. A total of 571 RA patients were involved in the analyses.Patient samples were compared to 305 healthy control samples also livingin the Leiden area. The protocols were approved by the local ethicscommittee and informed consent was obtained.

Detection of Anti-CarP Antibodies by ELISA

In brief, Non-modified FCS and modified-FCS were coated on NUNCMAXISORP™ plates (Thermo Scientific) over night. Following washings andblocking, the wells were incubated with serum. Bound human IgG or IgAwas detected using rabbit anti-human IgG or IgA antibodies (DAKO),followed by HRP-labeled goat anti-rabbit IgG antibody (Dako). Followingthe last washings, HRP enzyme activity was visualized using ABTS.⁽⁴¹⁾ Amore detailed description of the protein modifications and ELISA assaysbased on FCS and Fib, including F(ab)2, is available online(SI-materials and Methods). The cut-off for a positive response wasestablished as the mean plus 2× the standard deviation of the specificanti-CarP reactivity of the healthy controls. The methods for thedetection of ACPA and Western blotting are available online(SI-materials and Methods).

ELISA for Fib Peptides

Streptavidin (Invitrogen) was coated at 2 μg/ml in 100 μl on Nunc platesat 4° C. ON. After washing, Fib peptides containing either an arginine,citrulline, homocitrulline or a lysine (FIG. 8, Panel A)⁽⁴²⁾ wereincubated at 10 μg/ml in 100 μl PTB for 1 hour at RT. Next, thereactivity of antibodies reactive to these antigens was detected asdescribed above.

Inhibition Studies

To determine whether anti-CarP antibodies and ACPA are cross-reactiveantibodies, we performed inhibition studies in whichautoantibody-positive serum samples, positive for both ACPA andanti-CarP antibodies, were pre-incubated with increasing concentrationsof either non-modified FCS, Ca-FCS, Ci-FCS or the citrulline- orarginine-containing form of the CCP1 peptide.⁽⁴³⁾ Followingpre-incubation at room temperature (RT), the samples were tested forreactivity against Ca-FCS and Ci-FCS as described above. Serum andF(ab′)2 samples positive for both Ci-Fib and Ca-Fib were pre-incubatedwith Fib, Ci-Fib and Ca-Fib at 4° C. ON and subsequently analyzed on theFib ELISA (SI-materials and Methods).

Radiological Progression

In the EAC cohort, radiographs of the hands and feet, which had beenobtained in a longitudinal fashion, were scored according to theSharp/van der Heijde method.⁽⁴⁴⁾ Scoring and analysis have beendescribed in detail before.⁽²¹⁾ Data are analyzed directly, or usingrepeated measurement analysis, as to optimally make use of thelongitudinal data obtained for each patient.⁽²¹⁾ More detailedinformation is available online (SI-materials and Methods).

Generation of Antigens

Because it was not known whether antibodies against carbamylatedproteins would be present in sera of RA patients, or which proteins theywould recognize, a study of a diverse set of carbamylated proteins wascommenced in order to maximize the chances of detecting as many of theanti-CarP reactivities as possible. For this purpose, fetal calf serum(FCS) (Bodinco) that was carbamylated, citrullinated or left untreatedwas used. For generating carbamylated FCS (Ca-FCS), FCS was diluted inH₂O to 4 mg/ml and potassium cyanate (Sigma) was added to aconcentration of 1 M. Following incubation at 37° C. for 12 hours, thesample was extensively dialyzed against H₂O. Carbamylated fibrinogen(Ca-Fib) was generated by incubating 5 mg/ml fibrinogen (Fib) with 0.5 Mpotassium cyanate at 4° C. for three days, followed by extensivedialysis against PBS. Citrullinated FCS (Ci-FCS) and citrullinatedfibrinogen (Ci-Fib) was generated by incubation of 10 mg FCS or Fib in avolume of 1 mL containing 0.1 M Tris-HCl pH 7.6, 0.015 M CaCl₂ and 40 UPAD4 (Sigma) for 24 hours at 37° C. The presence of citrulline andhomocitrulline residues was confirmed using mass-spectrometry analysis.For Fib, extensive citrullination and complete carbamylation wasobserved in the protein segments analyzed.

Detection of Anti-CarP Antibodies by ELISA

Non-modified FCS and modified-FCS were coated at 10 μg/ml in 50 μl(diluted in pH 9.6 0.1 M carbonate-bicarbonate buffer) (CB) on NUNCMAXISORP™ plates (Thermo Scientific) overnight (ON). Following washingin PBS containing 0.05% TWEEN® (Sigma) (PT), the plates were blocked byincubating 100 μl PBS/1% bovine serum albumin (BSA) (Sigma) for 6 hoursat 4° C. Following additional washing, the wells were incubated with 50μl serum at a 1/50 dilution in PBS/0.05% TWEEN®/1% BSA buffer (PTB) onice overnight. All subsequent incubations are performed in PTB. As astandard, serial dilutions of a pool of positive sera were used. HumanIgG or IgA was detected using rabbit anti-human IgG antibody (DAKO) orrabbit anti-human IgA antibody (Dako) incubated on ice for 3.5 hours.Following washing, wells were incubated on ice for 3.5 hours withHRP-labeled goat anti-rabbit IgG antibody (Dako). Following the lastwashings, HRP enzyme activity was visualized using ABTS as describedbefore.⁽²²⁾ Sera of healthy subjects (n=305) were used as controls. Theabsorbance on both Ca-FCS and FCS was transformed to aU/mL andsubtracted the background signal (aU/mL) of FCS from the signal (aU/mL)of Ca-FCS as to analyze the specific anti-CarP reactivity (FIG. 7). Thecut-off was established for a positive response as the mean plus 2× thestandard deviation of the specific anti-CarP reactivity of the healthycontrols.

ELISA for Fibrinogen

Non-modified Fib Ci-Fib and Ca-Fib were coated at 20 μg/ml in 50 μl(diluted in pH 9.0 PBS) on NUNC MAXISORP™ plates ON. Following washingin PT, the plates were blocked by incubating 200 μl pH 9.0 PBS/2% BSAfor 2 hours at 4° C. Following additional washing, the wells wereincubated with 50 μl serum at a 1/50 dilution in RIA buffer (10 mM TrispH 7.6; 350 mM NaCl; 1% TritonX; 0.5% Na-deoxycholate; 0.1% SDS) (Sigma)on ice for 3 hours. All subsequent incubations are performed in RIAbuffer. As a standard, serial dilutions of a pool of positive sera wereused. Human IgG was detected using HRP-labeled rabbit anti-human IgGantibody (DAKO) incubated on ice for 2 hours. Following the lastwashings, HRP enzyme activity was visualized using ABTS. Sera of 214 RApatients and 54 healthy subjects as controls was analyzed. Theabsorbance on Fib Ci-Fib and Ca-Fib was transformed to aU/mL. Thecut-off was established for a positive response as the mean plus 2× thestandard deviation of the specific anti-CarP reactivity of the healthycontrols. These assays were repeated three times showing the same data.

F(ab′)2 Preparation

Total IgG from two anti-CarP-positive and two control sera were isolatedvia a HiTrap™ protein A HP column (GE Healthcare) following the protocolfor the column provided by the manufacturer. F(ab′)2 fragments weregenerated from purified IgG samples using a F(ab′)2 Preparation Kit(Thermo Scientific) following the protocol provided by the manufacturer.The molecular nature of the intact IgG and the F(ab′)2 was verifiedusing Coomassie-stained SDS page gels. These F(ab′)2 were used in ELISAas described above, now using either HRP-labeled rabbit anti-human IgG,IgA, IgM kappa, lamda antibody (anti-light chain) (Dako) or HRP-labeledrabbit anti-human IgG (Dako).

Detection of ACPA by ELISA

ACPA were measured by the CCP2 ELISA (Immunoscan RA Mark 2;Eurodiagnostica, Arnhem, The Netherlands). Samples with a value above 25units/ml were considered positive according to the manufacturer'sinstructions. A small percentage of ACPA-positive RA patients may beoutside the anti-CCP2 reactivity and, therefore, both terms will be usedto explicitly indicate what has been used in our analyses.

ACPA reactivity toward Ci-FCS was detected using ELISA plates that werecoated with Ci-FCS (50 μl/well 10 μg/ml) diluted with CB in the NUNCMAXISORP™ plates ON at 4° C. The plates were washed in PT followed byblocking with 100 μl PBS/1% BSA solution at 37° C. for 1 hour. Followingwashing, sera were incubated at a 1/50 dilution in 50 μl PTB andincubated at 37° C. for 1 hour. After washing, human IgA and IgG weredetected as described above.

Detection of Anti-CarP Antibodies by Western Blot

FCS, Ca-FCS and Ci-FCS were loaded onto 10% sodium dodecyl sulfate(SDS)-polyacrylamide gels and transferred onto Hybond-C Extra membranes(Amersham). Blots were incubated in blocking buffer (3% ELKMilk/PBS/0.05% TWEEN®) for 1 hour at RT, following washing with PT. Theblots were subsequently incubated with 2.5 ml 1:500 diluted serum inblocking buffer for 1.5 hours at RT. The sera were either ACPA-positiveanti-CarP negative or ACPA-negative anti-CarP positive as determined byELISA. After three washes with PT, blots were incubated with 5 mLHRP-labeled rabbit anti-human IgG diluted in blocking buffer for 1 hourat RT. Next, blots were washed and bound antibodies were visualizedusing enhanced chemiluminescence (Amersham).

Statistics of Radiological Progression

Association between anti-CarP antibodies positivity and radiographicprogression was analyzed using the Statistical Package for the SocialSciences (SPSS) 17.0 as described before. P-values below 0.05 wereconsidered statistically significant. A multivariate normal regressionanalysis for longitudinal data was used with radiological score asresponse variable. This method analyses repeated measurements at onceand takes advantage of the correlation between these measurements, whichresults in a more precise standard error. Radiological scores werelog-transformed to obtain a normal distribution. The rate of jointdestruction over time was tested by an interaction of time withanti-CarP. The effect of time was assumed to be linear in theinteraction term. The effect of time was entered as factor in the modelas well, allowing a mean response profile over time. Age, gender andinclusion period as proxy for treatment were included as correctionvariables in all analyses. In a separate analysis, the effect ofanti-CarP antibodies was corrected for the effect of anti-CCP and RF.

Example 1 Results Detection of Anti-CarP Antibodies

A novel ELISA was generated to detect anti-CarP antibodies from serumand synovial fluid using plates coated with, in vitro-generated,carbamylated FCS. A standard was generated from a pool of positive serashowing a dose-dependent binding of both IgG and IgA to Carbamylated FCS(Ca-FCS) and no binding to the native FCS (FIG. 1A). Sincehomocitrulline and citrulline are very similar residues but differ fromeach other by one atom, excluding the possibility that the anti-CarPantibodies were actually ACPA was desired. As a first test to excludethis, inhibition studies were performed that show that anti-CarPantibody binding to Ca-FCS can only be inhibited by Ca-FCS itself andnot by citrullinated FCS (Ci-FCS), native FCS or by peptides used todetect ACPA (FIG. 1B), indicating that anti-CarP is truly a differentreactivity. In addition, binding of ACPA to CCP2 plates was notinhibited by addition of Ca-FCS (data not shown).

Since these methods rely on ELISA, confirmation of those findings usingWestern blotting was desired. FCS and Ca-FCS was run on non-reducinggels, and following Western blotting, stained the blots using sera ofpatients that were positive or negative for anti-CarP antibodies asdetected by ELISA. Serum of anti-CarP antibody-positive patients testedpositive on lanes loaded with Ca-FCS, while no reactivity was seen inlanes loaded with FCS (FIG. 1C). Sera from anti-CarP antibody-negativepatients did not show such staining. Using Coomassie staining on loadedgels, it was confirmed that the FCS-loaded lanes did contain similarprotein amounts. Collectively, these data indicate that a novel assayhas been generated that can specifically detect anti-CarP antibodies,both IgG and IgA.

Anti-CarP Antibodies are Present in RA

From the Leiden Early Arthritis Clinic (EAC), patients suffering from UAor RA have been used according to the 1987 inclusion criteria. Inaddition, healthy controls were also used from the Leiden region. Thepresence of anti-CarP antibodies in patients and controls was measuredsimultaneously. OD values were calculated to arbitrary units per mLusing a standard. Healthy persons were used to calculate the cut-off forpositivity as defined by the mean plus 2× the standard deviation of thehealthy controls. Samples were considered to be positive when they had atiter higher than the cut-off and an absorbance that was at least 0.1units higher on Ca-FCS as compared to FCS.⁽¹⁷⁾ Using this approach, itwas established that 42% of the RA patients were positive for IgGanti-CarP antibodies, whereas 54% of sera from RA patients tested werepositive for IgA anti-CarP-antibodies (FIG. 2). Analysis of pairedserum/synovial fluid samples revealed that anti-CarP IgG and IgA canalso be found in synovial fluid of patients that are positive for theseautoantibodies in serum (data not shown).

Anti-CarP antibodies are present in serum and synovial fluid in asubstantial proportion of RA patients.

Anti-CarP Antibodies are Independent from ACPA

Next, it was analyzed as to whether the anti-CarP antibodies occurindependently of ACPA. To this end, the relationship between ACPA andanti-CarP antibodies in a set of 373 RA patients was analyzed. The datashow that 14% and 23% of the RA patients did not display ACPA but didharbor anti-CarP IgG and IgA, respectively. Likewise, 22% and 19% of theRA patients were positive for ACPA but negative for anti-CarP IgG andIgA, respectively (FIG. 3). Zooming in on the ACPA-negative individuals,it was observed that around 50% of all ACPA-negative RA patients testedpositively for anti-CarP antibodies. Thus, anti-CarP antibodies occurindependently from ACPA.

Anti-CarP Antibodies are Predictive for Development of RA

Patients presenting themselves at baseline with a diagnosis ofundifferentiated arthritis can go into remission, develop another formof arthritis or can develop RA. Clinically, it would be relevant to beable to discriminate between the patients in need for treatment and thepatients that will remit spontaneously. Therefore, 425 patients that hadUA at baseline for the presence of anti-CarP antibodies and thedevelopment of RA (n=151) were analyzed. It was observed that positivityfor IgG anti-CarP antibodies did not associate with RA developmentaccording to the 1987 criteria in a statistically significant manner(p=0.11). In contrast, IgA anti-CarP antibodies were strongly associatedwith future development of RA in the UA group as a whole (p=0.002) (FIG.4). This effect was especially prominent in the ACPA-negativeindividuals (FIG. 4).

Measuring anti-CarP antibodies in patients suffering fromundifferentiated arthritis is useful to identify persons at risk todevelop RA.

Anti-CarP Antibodies are Associated with More Severe Radiological Damage

Finally, analysis was performed to ascertain whether RA patients thatare positive at baseline for anti-CarP antibodies would have a differentclinical course of their disease. Therefore, comparisons were made as tothe extent of joint damage over time. Also in this analysis, positivityfor IgG anti-CarP antibodies did not associate with radiological damagein a statistically significant manner (p=0.43). However, IgA anti-CarPantibodies are strongly associated with more severe damage to the joints(p=0.002) (FIG. 5), an effect that was independent from RheumatoidFactor (RF) or ACPA. Together, these data indicate that anti-CarPantibodies are associated with a more severe disease course, independentof the presence of RA and or ACPA.

Discussion

A novel family of autoantibodies that recognize carbamylated proteins(anti-CarP) is described. These anti-CarP antibodies can be detected inboth the IgG and IgA isotypes. Both inhibition studies and cohortstudies show that anti-CarP antibodies are different from ACPA.Interestingly, positivity for anti-CarP, especially IgA, has clinicalimplications as individuals positive for anti-CarP IgA have an increasedrisk to progress from UA to RA and anti-CarP IgA-positive RA patientshave a worse outcome compared to anti-CarP IgA-negative RA patients.

A complex protein mix was used as an initial source of carbamylatedprotein antigens and, therefore, generated Ca-FCS. It was observed thatantibodies exist that are specifically directed against the carbamylatedform of FCS, which do not bind to native or citrullinated FCS in bothELISA and Western blot systems. These antibodies are of both the IgG andIgA isotypes, indicating that they are derived from class-switched Bcells, a process that would require T cell help. Indeed, data indicatethat homocitrulline directed T cells can be induced by immunization withcarbamylated model antigens.⁽¹¹⁾

It is shown herein that detection of these antibodies in early arthritiscan predict the future development of RA and predict a more severedisease course. Since it has been shown that early aggressive treatmentis beneficial,^((18, 19)) the invention provides methods for arthritistreatment of individuals suffering from, or at risk of suffering from,arthritis, the method comprising an arthritis diagnosis of theindividual wherein the diagnosis comprises a method for determining ananti-CarP antibody in a sample comprising a body fluid of theindividual. Preferably, the sample was determined to contain ananti-Carp antibody. A more stringent treatment of the anti-CarP-positiveindividual is beneficial to the patient.

In conclusion, next to the autoantibody system that recognizescitrullinated proteins (ACPA), an autoantibody system is also presentagainst carbamylated proteins (anti-CarP). Detection of such antibodiesis useful since its presence is, independently of ACPA, associated withdevelopment of UA to RA and is associated with a more severe diseasecourse.

Example 2 Results Anti-CarP Antibodies and ACPA are Different AntibodyFamilies

To detect antibodies against carbamylated proteins (anti-CarPantibodies), an ELISA was developed using carbamylated FCS (Ca-FCS) andnon-modified FCS as antigens. Analyzing sera of 40 RA patients and 40controls, it was observed that sera of RA patients reacted with Ca-FCSas compared to sera obtained from healthy subjects with both IgG (FIG.7, Panels A and B) and IgA (FIG. 7, Panels C and D) reactivity. Theenhanced reactivity of RA sera to Ca-FCS is further emphasized aftersubtraction of the reactivity against unmodified FCS (FIG. 7, Panels Cand E). Since citrulline and homocitrulline are two rather similar aminoacids (FIG. 6), it was next determined whether ACPA also recognizeshomocitrulline when located at the same position as citrulline in apeptide. For this purpose, ELISAs were performed using a citrullinatedFib peptide known to be recognized by ACPA.⁽²⁰⁾ Within this peptidebackbone, a citrulline, an arginine, a homocitrulline or a lysineresidue was introduced for further analysis. Analyzing a set of 76 RAsera, it was observed that ACPA only recognized the citrullinatedpeptide, but not the arginine-containing or thehomocitrulline-containing peptide (FIG. 8, Panel A). These data indicatethat ACPA can discriminate between citrulline and homocitrulline presentwithin the same peptide backbone. Next, analysis was performed as towhether there is cross-reactivity between anti-CarP antibodies and ACPAfor binding to post-translationally modified proteins. Therefore,inhibition studies were performed using sera that were reactive to bothcitrullinated and carbamylated antigens. The binding of anti-CarPantibodies to Ca-FCS-coated plates following pre-incubation with Ca-FCS,citrullinated FCS (Ci-FCS), native FCS or by citrullinated peptides usedto detect ACPA (CCP1) was analyzed. Following pre-incubation, it wasobserved that anti-CarP antibody binding to Ca-FCS can only be inhibitedby Ca-FCS but not by citrullinated FCS (Ci-FCS), native FCS or bypeptides used to detect ACPA (FIG. 8, Panel B). The reverse inhibitionexperiment was performed where the binding of ACPA to plates coated withCi-FCS following the same pre-incubation procedure was analyzed. It wasobserved that ACPA binding to Ci-FCS could only be inhibited by Ci-FCSand the citrullinated peptide but not by Ca-FCS, non-modified FCS, orthe arginine form of the peptide (FIG. 8, Panel C). Together, these dataindicate that anti-CarP antibodies and ACPA are not (or only limited)cross-reactive and specifically directed against homocitrulline,respectively, citrulline-containing antigens. Since all observationsdescribed above were made using ELISA, confirmation of the findingsusing a different technique was desirable. For this reason, a Westernblot analysis was performed using FCS, Ca-FCS and Ci-FCS on reducedgels, followed by Western blotting. The different blots were incubatedwith sera of individuals that were either anti-CarP positive and ACPAnegative or anti-CarP negative and ACPA positive. A positive staining ofthe anti-CarP-positive sample was observed only on Ca-FCS but not onCi-FCS or FCS (FIG. 8, Panel D). In contrast, the anti-CarP negative,ACPA-positive sample reacted to Ci-FCS, but not to Ca-FCS and FCS (FIG.8, Panel D). To confirm the presence of anti-CarP antibodies, theseexperiments were repeated using a more defined protein, human Fib, astarget antigen. Fib was citrullinated by PAD (Ci-Fib) or carbamylated bycyanate (Ca-Fib). The non-modified form (Fib), Ci-Fib and Ca-Fib wereused as antigens in ELISA. Similar to the observations for FCS,significant binding of antibodies to the Ci-Fib and the Ca-Fib but notto the Fib-coated wells was observed (FIG. 9, Panel A). This was largelyrestricted to the RA sera and not the controls (p=<0.0001). To analyzecross-reactivity, inhibition studies were performed as described above.ELISA analyses confirmed that ACPA and anti-CarP antibodies are largelynon-cross-reactive. To ensure that reactivity toward carbamylatedproteins is mediated by the antigen-binding-part of the antibodies,F(ab′)2 was generated. As expected, F(ab′)2, generated from anti-CarPIgG-positive samples but not from negative samples, display anti-CarPreactivity (FIG. 9, Panels C and D). As observed using intactantibodies, F(ab′)2 reactivity toward Ca-Fib could be inhibitedspecifically by Ca-Fib, whereas F(ab′)2 reactivity toward Ci-Fib couldonly be inhibited specifically by Ci-Fib (FIG. 9, Panel E).

Collectively, these data indicate that anti-CarP antibodies and ACPArecognize different antigens, one recognizing citrullinated proteins(ACPA) and the other carbamylated proteins (anti-CarP). Likewise, thesedata indicate that antigen recognition is most likely mediated via thevariable domains present in the F(ab′)2 fragments.

Anti-CarP Antibodies are Present in RA

Following the identification of anti-CarP antibodies as an autoantibodyfamily separate from ACPA, quantifying the presence of these anti-CarPantibodies in a large population of RA patients and controls wasdesired. For this reason, first, a standard was generated comprising apool of anti-CarP antibody-positive sera. This standard displayed aspecific, dose-dependent, binding of both IgG and IgA to carbamylatedFCS (Ca-FCS) but no binding to unmodified FCS (FIG. 10, Panels A and B).For this analysis, the FCS-based assay was again used in an attempt tocapture as many anti-CarP reactivities as possible. A cut-off forpositivity was established using sera of 305 healthy individuals asdescribed in the methods section. Using this approach, it was observedthat 45% of the sera of RA patients analyzed are positive for IgGanti-CarP antibodies (FIG. 10, Panel C). Likewise, 43% of sera from RApatients tested are positive for IgA anti-CarP-antibodies (FIG. 10,Panel D).

Anti-CarP Antibodies are Also Present in Sera of Anti-CCP2-negative RAPatients

The group of RA patients analyzed in this study consisted of bothACPA-positive and ACPA-negative individuals, as measured by the CCP2assay. Therefore, the association between anti-CarP antibodies andanti-CCP2 antibodies was analyzed next. The presence of anti-CarPantibodies and anti-CCP2 antibodies showed a limited degree ofcorrelation when analyzing the entire RA population (r²=0.27, p<0.001for anti-CarP IgG or r²=0.15, p<0.001 for IgA). However, substantialnumbers of RA patients were also identified that are only positive foranti-CCP2 antibodies as well as a group of patients that is onlypositive for anti-CarP antibodies (FIG. 10, Panels E and F). It wasobserved that approximately 16% of the anti-CCP2-negative RA patientsdisplayed anti-CarP IgG antibodies, whereas 30% of theanti-CCP2-negative RA patients tested positive for anti-CarP IgA (FIG.10, Panels G and H). These data indicate that the presence of anti-CarPantibodies overlaps with the occurrence of anti-CCP2 antibodies, butthat this overlap is not absolute as over 30% of the anti-CCP2-negativepatients harbor anti-CarP antibodies. In total, more than 35% of allanti-CCP2-negative patients have either anti-CarP IgG or IgA antibodies.

Anti-CarP Antibodies are Associated with More Severe Radiological Damage

The presence of ACPA is associated with a more severe clinical diseasecourse as measured by radiological damage. To analyze whether thepresence of anti-CarP antibodies are also predictive for a more severedisease course, the extent of joint damage over time betweenanti-CarP-positive and -negative patients participating in the LeidenEAC cohort was compared. This cohort is an inception cohort of patientswith recent-onset arthritis where X-rays of hands and feet are taken ofall RA patients at yearly intervals to assess radiological damage usingthe Sharp/van der Heijde method.⁽²¹⁾ It was observed that the presenceof anti-CarP IgG strongly associates with a more severe diseaseprogression. Patients positive for anti-CarP IgG had more jointdestruction over seven years than IgG-negative patients without (β=2.01,95% CI 1.68-2.40, p=8.68×10⁻¹⁴) or with correction of ACPA and RF(β=1.41, 95% CI 1.13-1.76, p=0.002) (FIG. 13). Anti-CarP IgA wasassociated with more joint destruction over seven years than anti-CarPIgA-negative patients without correction of ACPA and RF (β=1.21, 95% CI1.01-1.45, p=0.041) but not after correction (p=0.855) (FIG. 13). As theanalysis described above does not show whether anti-CarP antibodiespredict radiological progression in the anti-CCP2-negative,anti-CCP2-positive or both RA subgroups, a stratified analysis was nextperformed. Importantly, this analysis revealed that the presence ofanti-CarP IgG is associated with a more severe joint damage in theanti-CCP2-negative subgroup (β=1.86, 95% CI 1.41-2.66, p=1.8×10⁻⁵) (FIG.11). Likewise, a similar trend toward more joint damage over time wasobserved for anti-CCP2 negative patients tested positive for IgAanti-CarP antibodies (β=1.25, 95% CI 0.98-1.58, p=0.071) (FIG. 13). Incontrast, in the anti-CCP2-positive subgroup, which is alreadycharacterized by severe joint destruction, no additional increase wasobserved in individuals that also harbored anti-CarP antibodies (FIG.13). Together, these data indicate that the detection of anti-CarPantibodies at baseline is predictive for a more destructive diseasecourse in anti-CCP2-negative RA as measured by the Sharp/van der Heijdemethod.

Discussion

A family of autoantibodies that recognize carbamylated proteins,anti-CarP antibodies can be detected in sera of RA patients. Bothinhibition studies and cohort studies show that anti-CarP antibodies andACPA represent two different and independent autoantibody families, onerecognizing carbamylated proteins and the other citrullinated proteins.The data show that anti-CarP antibodies and ACPA are, by and large,non-cross-reactive, although no exclusion is made that somecross-reactivity exists at the population level as is also indicated inrecent data obtained in rabbits after vaccination with carbamylatedproteins.⁽¹⁴⁾ Interestingly, positivity for anti-CarP antibodies isrelated to clinical outcome as individuals positive for anti-CarP IgG,but negative for anti-CCP2 antibodies, have a more destructive diseasecourse as compared to anti-CarP IgG-negative RA patients.

It is currently unknown which proteins undergo post-translationalmodifications like carbamylation. Carbamylation is mediated by cyanate,which is in equilibrium with urea. Increased urea concentrations,smoking and inflammation have been reported to shift this equilibriumtoward cyanate and, hence, enhanced carbamylation.⁽¹³⁾ Since currentlyno in vivo-relevant targets for anti-CarP antibodies are known, acomplex protein mixture was used as an initial source of carbamylatedprotein antigens for the detection of anti-CarP antibodies. Western blotanalyses indicate the recognition by anti-CarP antibodies of at leastone dominant protein present in FCS after carbamylation employingcyanate (representing high urea concentrations) (FIG. 8, Panel D).However, these data are likely not to represent the in vivo situationwhere carbamylation is a more gradual, but constantly occurringprocess.⁽²²⁾ In this respect, it is likely that especially long-livedproteins acquire homocitrulline residues over time as carbamylation isnearly irreversible and, thus, will lead to the accumulation ofhomocitrulline residues on proteins with a long half-life. Intriguingly,the joint is known for the presence of long-lived proteins such ascollagens and other cartilage-expressed proteins. Therefore, it isconceivable that such matrix-proteins will accumulate homocitrullineresidues during life, especially under conditions of inflammation.Indeed, it has been shown that homocitrulline is present in thejoint,⁽¹¹⁾ possibly representing the long-lived nature of manyjoint-derived proteins. It will be interesting to know the identity ofthese proteins and whether these can serve as a target for anti-CarPantibodies.

The molecular nature of the antigens recognized by ACPA was identifiedmore than 15 years ago by describing that citrulline is an essentialconstituent of antigens recognized by these RA-specificantibodies.^((23, 24)) This finding has made considerable impact as ithas opened up the way to relevant and novel insights into RA diagnosisand etiopathology.⁽¹⁾ For example, ACPA are now part of the newACR/EULAR criteria for RA,⁽²⁵⁾ and have been implicated in RApathogenesis, both in animal models^((26, 27, 28)) and in ex vivo humanstudies.^((29, 30, 31, 32)) Importantly, the description of ACPA has ledto the realization that RA constitutes at least two clinical syndromesthat share many clinical features, but differ with respect to geneticbackground, predisposing environmental factors and clinicalprogression/remission.^((33, 3, 4, 34, 35)) Although it is clearly tooearly to allow any firm conclusions, it is tempting to speculate thatanti-CarP antibodies also contribute to disease pathogenesis and/ordisplay diagnostic value, given the similar nature of the antigensrecognized and their presence in ACPA-negative disease.

The presence of anti-CarP antibodies in anti-CCP2-negative disease ishighly intriguing as it could potentially represent a novel biomarkerthat positively identifies at least part of this manifestation of RA. Togain further insight into this possibility, it is important to establishwhether the presence of anti-CarP antibodies is specific for RA or alsofound in other rheumatic diseases, as well as whether their presencepredict the development of (ACPA-negative) RA in patients suffering fromearly unclassified RA and/or joint complaints such as arthralgia.

To establish a cut-off to define a positive sample, the presence of IgGand IgA directed against Ca-FCS and FCS in sera of healthy controls wasanalyzed. All samples were tested for reactivity toward Ca-FCS and FCS,and absorbance values were converted into aU/mL using an anti-CarPantibody-positive standard present on the same plate. Since sera fromseveral individual subjects also displayed reactivity towardnon-modified FCS, the “FCS reactivity” was subtracted from thereactivity toward Ca-FCS using aU/mL as defined by the standard curve.Subsequently, the cut-off was calculated as the mean plus two timesstandard deviation and applied the cut-off to the data of the RApatients following a similar strategy. The disadvantage of this methodis that a standard is used on Ca-FCS for the determination of aU/mLtoward FCS, another antigenic entity. However, this method did allow thecalculation of a specific response to the post-translationalmodification.

Every method of establishing a cut-off has advantages and limitations.Therefore, the observations were subsequently confirmed using anotherstrategy as well, by calculating the cut-off as the mean plus two timesstandard deviation of the anti-Ca-FCS response in controls. This cut-offwas applied to the data of the RA patients as was also employedbefore.⁽³⁶⁾ The association with radiological progression of IgG inACPA-negative RA remains significant, albeit with a lower level ofsignificance (p=0.001).

From a clinical perspective, the detection of anti-CarP antibodies inearly arthritis could be highly rewarding since they predict a moresevere disease course. Since early aggressive treatment in RA has beenshown to prevent future damage,^((37, 38)) the detection of anti-CarPantibodies might be beneficial to identify anti-CCP2-negative patientsat risk to develop severe disease. The identification of such patientsmight be important to guide treatment decisions early after onset ofsymptoms, especially in early arthritis patients that are difficult toclassify.

In conclusion, in addition to the autoantibody system that recognizescitrullinated proteins (ACPA), an autoantibody system againstcarbamylated proteins (anti-CarP) is present in sera of RA patients.Detection of anti-CarP antibodies could offer new possibilities toidentify patients at risk for a severe disease course.

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TABLE I  List of lysine-containing peptides of fibrinogen alphaand their homocitrulline-containing counterpartsLysine-containing peptides Homocitrulline-containing peptidesof fibrinogen alpha of fibrinogen alpha SEQ ID SEQ ID NO: NO: 1 4RVVERHQSACKDSDWPFCSDE 1 5 RVVERHQSAChomocitDSDWPFCSDE 2 6PFCSDEDWNYKCPSGCRMKGL 2 7 PFCSDEDWNYhomocitCPSGCRMhomocitGL 3 8NYKCPSGCRMKGLIDEVNQDF 3 9 NYhomocitCPSGCRMhomocitGLIDEVNQDF 4 10VNQDFTNRINKLKNSLFEYQK 4 11 VNQDFTNRINhomocitLhomocitNSLFEYQhomocit 5 12QDFTNRINKLKNSLFEYQKNN 5 13 QDFTNRINhomocitLhomocitNSLFEYQhomocitNN 6 14KLKNSLFEYQKNNKDSHSLTT 6 15 homocitLhomocitNSLFEYQhomocitNNhomocitDSHSLTT7 16 NSLFEYQKNNKDSHSLTTNIM 7 17 NSLFEYQhomocitNNhomocitDSHSLTTNIM 8 18EDLRSRIEVLKRKVIEKVQHI 8 19 EDLRSRIEVLhomocitRhomocitVIEhomocitVQHI 9 20LRSRIEVLKRKVIEKVQHIQL 9 21 LRSRIEVLhomocitRhomocitVIEhomocitVQHIQL 10 22IEVLKRKVIEKVQHIQLLQKN 10 23IEVLhomocitRhomocitVIEhomocitVQHIQLLQhomocitN 11 24EKVQHIQLLQKNVRAQLVDMK 11 25 EhomocitVQHIQLLQhomocitNVRAQLVDMhomocit 1226 KNVRAQLVDMKRLEVDIDIKI 12 27 homocitNVRAQLVDMhomocitRLEVDIDIhomocitI13 28 MKRLEVDIDIKIRSCRGSCSR 13 29 MhomocitRLEVDIDIhomocitIRSCRGSCSR 1430 SRALAREVDLKDYEDQQKQLE 14 31 SRALAREVDLhomocitDYEDQQhomocitQLE 15 32VDLKDYEDQQKQLEQVIAKDL 15 33 VDLhomocitDYEDQQhomocitQLEQVIAhomocitDL 1634 QQKQLEQVIAKDLLPSRDRQH 16 35 QQhomocitQLEQVIAhomocitDLLPSRDRQH 17 36SRDRQHLPLIKMKPVPDLVPG 17 37 SRDRQHLPLIhomocitMhomocitPVPDLVPG 18 38DRQHLPLIKMKPVPDLVPGNF 18 39 DRQHLPLIhomocitMhomocitPVPDLVPGNF 19 40PVPDLVPGNFKSQLQKVPPEW 19 41 PVPDLVPGNFhomocitSQLQhomocitVPPEW 20 42VPGNFKSQLQKVPPEWKALTD 20 43 VPGNFhomocitSQLQhomocitVPPEWhomocitALTD 2144 SQLQKVPPEWKALTDMPQMRM 21 45 SQLQhomocitVPPEWhomocitALTDMPQMRM 22 46SSGTGGTATWKPGSSGPGSTG 22 47 SSGTGGTATWhomocitPGSSGPGSTG 23 48PGTRREYHIEKLVTSKGDKEL 23 49 PGTRREYHTEhomocitLVTShomocitGDhomocitEL 2450 EYHIEKLVTSKGDKELRTGKE 24 51EYHTEhomocitLVTShomocitGDhomocitELRTGhomocitE 25 52TEKLVTSKGDKELRTGKEKVT 25 53 TEhomocitLVTShomocitGDhomocitELRTGhomocitEhomocitVT 26 54 SKGDKELRTGKEKVTSGSTTT 26 55ShomocitGDhomocitELRTGhomocitEhomocitVTSGSTTT 27 56GDKELRTGKEKVTSGSTTTTR 27 57 GDhomocitELRTGhomocitEhomocitVTSGSTTTTR 2858 STTTTRRSCSKTVTKTVIGPD 28 59 STTTTRRSCShomocitTVThomocitTVIGPD 29 60TRRSCSKTVTKTVIGPDGHKE 29 61 TRRSCShomocitTVThomocitTVIGPDGHhomocitE 3062 TKTVIGPDGHKEVTKEVVTSE 30 63 ThomocitTVIGPDGHhomocitEVThomocitEVVTSE31 64 IGPDGHKEVTKEVVTSEDGSD 31 65 IGPDGHhomocitEVThomocitEVVTSEDGSD 3266 AAFFDTASTGKTFPGFFSPML 32 67 AAFFDTASTGhomocitTFPGFFSPML 33 68GSESGIFTNTKESSSHHPGIA 33 69 GSESGIFTNThomocitESSSHHPGIA 34 70PGIAEFPSRGKSSSYSKQFTS 34 71 PGIAEFPSRGhomocitSSSYShomocitQFTS 35 72PSRGKSSSYSKQFTSSTSYNR 35 73 PSRGhomocitSSSYShomocitQFTSSTSYNR 36 74YNRGDSTFESKSYKMADEAGS 36 75 YNRGDSTFEShomocitSYhomocitMADEAGS 37 76GDSTFESKSYKMADEAGSEAD 37 77 GDSTFEShomocitSYhomocitMADEAGSEAD 38 78EADHEGTHSTKRGHAKSRPVR 38 79 EADHEGTHSThomocitRGHAhomocitSRPVR 39 80GTHSTKRGHAKSRPVRDCDDV 39 81 GTHSThomocitRGHAhomocitSRPVRDCDDV 40 82SGTQSGIFNIKLPGSSKIFSV 40 83 SGTQSGIFNIhomocitLPGSShomocitIFSV 41 84IFNIKLPGSSKIFSVYCDQET 41 85 IFNIhomocitLPGSShomocitIFSVYCDQET 42 86LNFNRTWQDYKRGFGSLNDEG 42 87 LNFNRTWQDYhomocitRGFGSLNDEG 43 88VRGIHTSPLGKPSLSP 43 89 VRGIHTSPLGhomocitPSLSP

TABLE II  List of lysine-containing peptides of fibrinogen betaand their homocitrulline-containing counterpartsLysine-containing peptides Homocitrulline-containing peptidesof fibrinogen beta of fibrinogen beta SEQ SEQ ID ID NO: NO: 1 90MKRMVSWSFHKL 1 91 MhomocitRMVSWSFHhomocitL 2 92 MKRMVSWSFHKLKTMKHLLLL 293 MhomocitRMVSWSFHhomocitLhomocitTMhomocitHLLLL 3 94RMVSWSFHKLKTMKHLLLLLL 3 95 RMVSWSFHhomocitLhomocitTMhomocitHLLLLLL 4 96SWSFHKLKTMKHLLLLLLCVF 4 97 SWSFHhomocitLhomocitTMhomocitHLLLLLLCVF 5 98LLLLLCVFLVKSQGVNDNEEG 5 99 LLLLLCVFLVhomocitSQGVNDNEEG 6 100FSARGHRPLDKKREEAPSLRP 6 101 FSARGHRPLDhomocithomocitREEAPSLRP 7 102SARGHRPLDKKREEAPSLRPA 7 103 SARGHRPLDhomocithomocitREEAPSLRPA 8 104SGGGYRARPAKAAATQKKVER 8 105 SGGGYRARPAhomocitAAATQhomocithomocitVER 9106 ARPAKAAATQKKVERKAPDAG 9 107ARPAhomocitAAATQhomocithomocitVERhomocitAPDAG 10 108RPAKAAATQKKVERKAPDAGG 10 109RPAhomocitAAATQhomocithomocitVERhomocitAPDAGG 11 110AAATQKKVERKAPDAGGCLHA 11 111 AAATQhomocithomocitVERhomocitAPDAGGCLHA 12112 SSSFQYMYLLKDLWQKRQKQV 12 113 SSSFQYMYLLhomocitDLWQhomocitRQhomocitQV13 114 YMYLLKDLWQKRQKQVKDNEN 13 115YMYLLhomocitDLWQhomocitRQhomocitQVhomocitDNEN 14 116LLKDLWQKRQKQVKDNENVVN 14 117LLhomocitDLWQhomocitRQhomocitQVhomocitDNENVVN 15 118DLWQKRQKQVKDNENVVNEYS 15 119 DLWQhomocitRQhomocitQVhomocitDNENVVNEYS 20120 PVVSCEEIIRKGGETSEMYLI 20 121 PVVSCEEIIRhomocitGGETSEMYLI 21 122MYLIQPDSSVKPYRVYCDMNT 21 123 MYLIQPDSSVhomocitPYRVYCDMNT 22 124VDFGRKWDPYKQGFGNVATNT 22 125 VDFGRhomocitWDPYhomocitQGFGNVATNT 23 126FGNVATNTDGKNYCGLPGEYW 23 127 FGNVATNTDGhomocitNYCGLPGEYW 24 128LPGEYWLGNDKISQLTRMGPT 24 129 LPGEYWLGNDhomocitISQLTRMGPT 25 130TELLIEMEDWKGDKVKAHYGG 25 131 TELLIEMEDWhomocitGDhomocitVhomocitAHYGG 26132 LIEMEDWKGDKVKAHYGGFTV 26 133 LIEMEDWhomocitGDhomocitVhomocitAHYGGFTV27 134 EMEDWKGDKVKAHYGGFTVQN 27 135EMEDWhomocitGDhomocitVhomocitAHYGGFTVQN 28 136 GGFTVQNEANKYQISVNKYRG 28137 GGFTVQNEANhomocitYQISVNhomocitYRG 29 138 EANKYQISVNKYRGTAGNALM 29139 EANhomocitYQISVNhomocitYRGTAGNALM 30 140 NDGWLTSDPRKQCSKEDGGGW 30141 NDGWLTSDPRhomocitQCShomocitEDGGGW 31 142 LTSDPRKQCSKEDGGGWWYNR 31143 LTSDPRhomocitQCShomocitED GGGWWYNR 32 144 WGGQYTWDMAKHGTDDGVVWM 32145 WGGQYTWDMAhomocitHGTDDGVVWM 33 146 TDDGVVWMNWKGSWYSMRKMS 33 147TDDGVVWMNWhomocitGSWYSMRhomocitMS 34 148 NWKGSWYSMRKMSMKIRPFFQ 34 149NWhomocitGSWYSMRhomocitMSMhomocitIRPFFQ 35 150 SWYSMRKMSMKIRPFFPQQ 35151 SWYSMRhomocitMSMhomocitIRPFFPQQ

TABLE III  List of lysine-containing peptides of fibrinogen gammaand their homocitrulline-containing counterpartsLysine-containing peptides Homocitrulline-containing peptidesof fibrinogen gamma of fibrinogen gamma SEQ ID SEQ ID NO: NO: 1 152IADFLSTYQTKVDKDLQSLED 1 153 IADFLSTYQThomocitVDhomocitDLQSLED 2 154FLSTYQTKVDKDLQSLEDILH 2 155 FLSTYQThomocitVDhomocitDLQSLEDILH 3 156LEDILHQVENKTSEVKQLIKA 3 157 LEDILHQVENhomocitTSEVhomocitQLIhomocitA 4158 HQVENKTSEVKQLIKAIQLTY 4 159 HQVENhomocitTSEVhomocitQLIhomocitAIQLTY5 160 NKTSEVKQLIKAIQLTYNPDE 5 161NhomocitTSEVhomocitQLIhomocitAIQLTYNPDE 6 162 QLTYNPDESSKPNMIDAATLK 6163 QLTYNPDESShomocitPNMIDAATLhomocit 7 164 KPNMIDAATLKSRKMLEEIMK 7 165homocitPNMIDAATLhomocitSRhomocitMLEEIMhomocit 8 166MIDAATLKSRKMLEEIMKYEA 8 167 MIDAATLhomocitSRhomocitMLEEIMhomocitYEA 9168 KSRKMLEEIMKYEASILTHDS 9 169 homocitSRhomocitMLEEIMhomocitYEASILTHDS10 170 LQEIYNSNNQKIVNLKEKVAQ 10 171LQEIYNSNNQhomocitIVNLhomocitEhomocitVAQ 11 172 NSNNQKIVNLKEKVAQLEAQC 11173 NSNNQhomocitIVNLhomocitEhomocitVAQLEAQC 12 174 NNQKIVNLKEKVAQLEAQCQE12 175 NNQhomocitIVNLhomocitEhomocitVAQLEAQCQE 13 176QLEAQCQEPCKDTVQIHDITG 13 177 QLEAQCQEPChomocitDTVQIHDITG 14 178DTVQIHDITGKDCQDIANKGA 14 179 DTVQIHDITGhomocitDCQDIANhomocitGA 15 180TGKDCQDIANKGAKQSGLYFI 15 181 TGhomocitDCQDIANhomocitGAhomocitQSGLYFI 16182 DCQDIANKGAKQSGLYFIKPL 16 183 DCQDIANhomocitGAhomocitQSGLYFIhomocitPL17 184 GAKQSGLYFIKPLKANQQFLV 17 185GAhomocitQSGLYFIhomocitPLhomocitANQQFLV 18 186 GSGNGWTVFQKRLDGSVDFKK 18187 GSGNGWTVFQhomocitRLDGSVDFhomocithomocit 19 188 QKRLDGSVDFKKNWIQYKEGF19 189 QhomocitRLDGSVDFhomocithomocitNWIQYhomocitEGF 20 190KRLDGSVDFKKNWIQYKEGFG 20 191homocitRLDGSVDFhomocithomocitNWIQYhomocitEGFG 21 192VDFKKNWIQYKEGFGHLSPTG 21 193 VDFhomocithomocitNWIQYhomocitEGFGHLSPTG 22194 GTIEFWLGNEKIHLISTQSAI 22 195 GTTEFWLGNEhomocitIHLISTQSAI 23 196RTSTADYAMFKVGPEADKYRL 23 197 RTSTADYAMFhomocitVGPEADhomocitYRL 24 198AMFKVGPEADKYRLTYAYFAG 24 199 AMFhomocitVGPEADhomocitYRLTYAYFAG 25 200GFDFGDDPSDKFFTSHNGMQF 25 201 GFDFGDDPSD homocitFFTSHNGMQF 26 202QFSTWDNDNDKFEGNCAEQDG 26 203 QFSTWDNDNDhomocitFEGNCAEQDG 27 204EQDGSGWWMNKCHAGHLNGVY 27 205 EQDGSGWWMNhomocitCHAGHLNGVY 28 206GVYYQGGTYSKASTPNGYDNG 28 207 GVYYQGGTYShomocitASTPNGYDNG 29 208YDNGIIWATWKTRWYSMKKTT 29 209 YDNGIIWATWhomocitTRWYSMhomocithomocitTT 30210 ATWKTRWYSMKKTTMKIIPFN 30 211ATWhomocitTRWYSMhomocithomocitTTMhomocitIIPFN 31 212TWKTRWYSMKKTTMKIIPFNR 31 213TWhomocitTRWYSMhomocithomocitTTMhomocitIIPFNR 32 214RWYSMKKTTMKIIPFNRLTIG 32 215 RWYSMhomocithomocitTTMhomocitIIPFNRLTIG

What is claimed is:
 1. A method for classifying an individual that issuffering from or at risk of suffering from a form of arthritis, themethod comprising: determining whether a sample comprising a body fluidof the individual comprises an anti-carbamylated protein (“anti-Carl'”)antibody.
 2. A method for providing a prognosis for the development ofarthritis to an individual suffering from the arthritis, the methodcomprising: determining whether a sample comprising a body fluid of theindividual comprises an anti-carbamylated protein (“anti-CarP”)antibody, and estimating the future severity of the arthritis based onthe detection of the anti-Carp antibody in the sample.
 3. The methodaccording to claim 1, wherein the body fluid is a serum sample or asynovial fluid sample.
 4. The method according to claim 1, wherein theanti-CarP is of the Ig-subtype IgA or of the Ig-subtype IgG.
 5. Themethod according to claim 1, wherein the method further comprises:determining whether a sample comprising a body fluid of the individualcomprises an anti-citrullinated protein antibody (“ACPA”).
 6. The methodaccording to claim 1, for determining whether the individual is at riskof suffering from arthritis, and wherein the individual was notsuffering from arthritis at the time the fluid sample was obtained. 7.The method according to claim 1, wherein the arthritis is rheumatoidarthritis, juvenile arthritis, psoriatic arthritis, osteoarthritis,polymyalgia rheumatica, ankylosing spondylitis, reactive arthritis,gout, pseudogout, autoimmune arthritis, systemic lupus erythematosus,polymyositis, fibromyalgia, Lyme disease, undifferentiated arthritis,non-rheumatoid arthritis, or spondyloarthropathy.
 8. The methodaccording to claim 7, wherein the arthritis is rheumatoid arthritis,juvenile arthritis, or undifferentiated arthritis.
 9. A kit fordetecting anti-CarP antibodies in a body fluid of an individual, the kitcomprising: a carbamylated protein or peptide.
 10. The kit of claim 9,further comprising: an anti-human IgG antibody and/or an anti-human IgAantibody.
 11. The kit of claim 10, wherein the anti-human Ig antibodycomprises an anti-human IgA antibody.
 12. The kit of claim 9, furthercomprising a citrullinated protein or peptide.
 13. The method accordingto claim 1, wherein the anti-Carp antibody is specific for acarbamylated protein or peptides derived from fetal calf's serum(“FCS”).
 14. The method according to claim 1, further comprisingdetermining a further factor as an arthritis classifier for theindividual.
 15. The method according to claim 14, wherein the furtherfactor comprises determining ACPA, rheumatoid factor, C-reactiveprotein, and/or erythrocyte sedimentation rate.
 16. The method accordingto claim 1, wherein the anti-CarP antibody is an anti-carbamylatedfibrinogen (“anti-Ca-Fib”) antibody.
 17. The method according to claim1, wherein the anti-CarP antibody is an anti-carbamylated fibrinogen(“anti-Ca-Fib”) antibody.
 18. The kit of claim 9, wherein the anti-CarPantibodies are an anti-carbamylated fibrinogen (“anti-Ca-Fib”).
 19. Thekit of claim 9, wherein the carbamylated protein or peptide is acarbamylated fibrinogen or a peptide derived therefrom.
 20. The methodaccording to claim 20, wherein the body fluid is a serum sample or asynovial fluid sample.